Philip J. Fialkow

Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage.

Glucose-6-phosphate dehydrogenase (G-6-PD) isoenzymes types of granulocytes were determined in eight women with chronic myelocytic leukemia (CML). The patients were heterozygous at the X-linked G-6-PD locus for the common gene, GdB, and a variant, such as GdA, so that both B and A enzyme types were found in skin cells. In contrast to these normal cells, only one G-6-PD type was found in CML granulocytes. The fact that such single-enzyme phenotypes are found in CML granulocytes, but not in nonleukemic granulocytes, provides strong evidence that the disease has a clonal origin. Single-enzyme phenotypes were also found in erythrocytes, platelets and cultured blood macrophages indicating that these cells have a common stem cell which is the site of the abnormality in CML. In the one studied patient, no evidence was found for involvement of cultured marrow fibroblasts. Clonal origin of CML virtually excludes cell recruitment as a sole pathogenetic mechanism. Either the leukemia arises as a consequence of a rare initial event in a single cell, or a series of events occurs in a clone such that it evolves into CML, or both.

Clonal Development, Stem-Cell Differentiation, and Clinical Remissions in Acute Nonlymphocytic Leukemia

To determine whether acute nonlymphocytic leukemia develops clonally, to study the pattern of differentiation of the involved stem cells, and to determine whether clinical remissions are true remissions, we studied 27 patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked glucose-6-phosphate dehydrogenase. In each case, leukemic blast cells manifested only one type of glucose-6-phosphate dehydrogenase, indicating that the malignant process had developed from a single cell. In six elderly patients, circulating erythrocytes, platelets, or both expressed only the glucose-6-phosphate dehydrogenase found in blast cells, indicating that these leukemias had arisen from stem cells with multipotent differentiative expression. In 16 younger adults and children, erythroid cells and platelets were predominantly derived from normal stem cells. In three other cases, the stem cell that gave rise to leukemic blasts apparently also gave rise to erythroid progenitors but not to mature erythrocytes. Heterogeneity was also found during remissions. In 8 of 13 patients, restoration of nonclonal hemopoiesis and repopulation of the marrow by normal stem cells was observed during remission. In the other five patients, marrow stem cells remained partially or completely clonal, even during remission. These data indicate that acute nonlymphocytic leukemia is a heterogeneous disease with respect to differentiation of the stem cells involved by leukemia and the nature of remissions.

The Origin and Development of Human Tumors Studied with Cell Markers

AMONG the more remarkable but least understood homeostatic mechanisms of higher organisms are those that govern tissue development, growth and repair. Considering the trillions of cells involved an...

Primordial cell pool size and lineage relationships of five human cell types

Several aspects of normal and neoplastic development can be studied in individuals who have two or more genetically distinct types of cells. Females heterozygous at the X-linked glucose-6phosphate dehydrogenase (G-6-PD) locus are cellular mosaics by virtue of fixed inactivation of one X chromosome in each somatic cell early in embryogenesis (Lyon, 1968). Thus, a given somatic cell in a GdBIGdA heterozygote has either GdB or GdA active (Beutler, Yeh & Fairbanks, 1962; Davidson, Nitowsky & Childs, 1963; DeMars & Nance, 1964). Clones derived from cells with an active GdA allele can be distinguished from those in which GdB is active by subjecting the respective enzymes to starch-gel electrophoresis the A type migrates more rapidly than the B enzyme. Therefore G-6-PD heterozygotes have two cell populations each of which is marked or tagged by the type of enzyme it produces. Since the first report by Linder & Gartler (1965) this system has been used extensively to trace the origin and development of human neoplasms (reviewed in Fialkow, 1972). A tumour with a clonal origin in a GdB/GdA heterozygote should contain only type B or type A enzyme (depending upon whether GdB or GdA was active in the single progenitor cell) ; a tumour with multicellular origin may contain both A and B enzymes. Similarly, the G-6-PD method can also be used to trace normal development. One application is to compare the ratio of A to B enzyme in the same organ from many subjects. Since the enzyme composition reflects the mosaic cell composition, with certain assumptions estimates can then be made of how many cells gave rise to that organ during embryogenesis. If it were derived from a single cell after X inactivation has occurred, only one type of enzyme, A or B, should be found. If there were two progenitor cells, the organs from 114 of heterozygotes should type as A, 112 a,s AB, and 114 as B, and so on. If the number of primordial cells is large, the organ from almost a.11 GdB/GdA females should contain approximately equal amounts of A and B enzyme. Another application study of many different tissues from a single subject provides information about cell lineage relationships. If two tissues are derived from a common progenitor pool they should show correlations in the ratio of the two isoenzymes. For example, Gandini & Gartler (1969) found excellent correlations among the ratios of A to B enzymes in red cells, granulocytes and blood lymphocytes and concluded that these cell lines are derived from a common stem cell pool. In previous reports the mosaic compositions of blood cells have been compared with that of skin in only two patients (Gandini et al. 1968). We have studied five human cell types red cells, granulocytes, blood lymphocytes, lymph nodes, skin a.nd muscle from 42 GdB/GdA heterozygotes.

Liver cysts in patients with autosomal dominant polycystic kidney disease

Liver cysts were found in 46 (29 per cent) of 158 patients over 10 years of age with documented autosomal dominant-type polycystic kidney disease (PKD) from 62 unrelated families. Hepatic cysts were not found in any patient at risk for PKD in whom renal cysts were not detected. The prevalence of liver cysts increased with advancing age and with declining rate of glomerular filtration. Results of clinical and laboratory studies indicate that polycystic liver disease in patients with autosomal dominant-type PKD is a benign condition, rarely, if ever, causing impaired liver function or portal hypertension.

Expression of the gene defect in X-linked agammaglobulinemia.

Although X-linked agammaglobulinemia was one of the first immunodeficiencies described,1 the genetic defect responsible for this disorder has not yet been identified. X-linked agammaglobulinemia is...

Acute nonlymphocytic leukemia: expression in cells restricted to granulocytic and monocytic differentiation.

Two patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase were studied to determine the number and type of cells in which the disease arises. Both type A and B isoenzymes were found in normal tissues, but the myeloblasts showed only one enzyme type, indicating that at the time of study, the disease had a clonal origin. The observation in one patient that erythroid cells did not arise from this clone contrasts with conclusions reached in patients previously studied with chromosomal markers. The results suggest that in this patient, the leukemic clone suppressed expression of normal granulopoiesis but did not inhibit erythroid differentiation from normal progenitors. They suggest also that the disease is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-macrophage pathway; in others, it involves stem cells that also differentiate into erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic importance.

Polycythemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin.

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.

Multiple Cell Origin of Hereditary Neurofibromas

Abstract In a female heterozygous for the A and B genes at the X-linked glucose-6-phosphate dehydrogenase (G-6-PD) locus, single cells express the activity of only one or the other gene. Thus, this...

The use of cell markers in the study of human hematopoietic neoplasia.

Publisher Summary This chapter summarizes the major findings that are significant in understanding the clonal development and progression of human hemopoietic neoplasia. Determination of whether a cellular proliferation is monoclonal or polyclonal has important pathogenetic implications. Polyclonal proliferations are often the result of normal processes in which a tissue responds appropriately to an exogenous stimulus. On the other hand, monoclonal proliferations reflect disordered hemopoiesis in which a clone of cells gains in vivo proliferative advantage. The ability to detect monoclonality in proliferating cells requires a marker system that enables the progeny of different cells to be recognized. Markers that are used effectively to investigate human lymphoid neoplasia include immunoglobulin (Ig) proteins synthesized by relatively mature cells of B-lymphoid origin, DNA sequences coding for these Igs, and T-cell receptor gene rearrangements. Beyond determining whether a disease state is clonal at the time of presentation, studies with cell markers allow characterization of the clonal state throughout subsequent stages of the disease and the differentiative expression of the stem cell involved by the clonal proliferation. Many human hemopoietic neoplasias appear to have a multistep pathogenesis. The diploid cell may represent an early stage in leukemogenesis. Subsequent development of structural chromosomal alterations or aneuploidies may lead to frank leukemia by any of a number of mechanisms, such as activation of an oncogene, amplification of a gene whose product is important for growth, or loss of a gene involved in repressing or regulating growth. The malignant phenotype then progresses by stepwise evolution.